Process for splitting racemates



United States Patent 2,833,694 PROCESS FOR ISPLITTING RACEMATES Vladimir Prelog, Zurich, Switzerland, assigns! to Ciba Pharmaceutical Products, Inc., Summit, N. J.

No Drawing. Application August 16, 1956 Serial No. 604,341

Claims priority, application Switzerland August 25, 1955 26 Claims. (Cl. 195-2) In copending application Serial No. 604,334, filed by A. Wettstein et al., filed August 16, 1956, there is described a process in which by the action of reducing enzymes of micro-organisms upon d, l-steroids essentially only the natural d-form is reduced and the l-form remains unchanged. By means of this process it is accordingly possible to split racemic steroid compounds into the antipodes, the d-antipode being at the same time converted, viz. reduced.

I have now found a new process for splitting racemates. In this process racemates of bicyclic-alicyclic compounds which contain at least one grouping reducible with the formation of at least one new centre of asymmetry are subjected to the action of reducing enzymes produced by cultures of micro-organisms and at least one of the resulting optically active products is isolated and, if desired any hydrogenation product obtained is treated with a dehydro-genating agent.

The starting materials contain as reducible groupings for example carbon-oxygen and/ or carbon-carbon double bonds. They belong especially to the hydronaphthalene and the hydro-indene series, for example to the dihydro-, tetrahydro-, hexahydro-, octahydroor decahydro-naphthalene series, or to the dihydro-, tetrahydro-, hexahydroor octahydro-indene series. They can be substituted in any desired manner. As substitnents they are concerned especially free or functionally converted hydroxy1-, oxoor carboxylgroups, such as ester, amide, nitrile, ether, thioester, thioether, thioland thione-ester, acetal, mercaptal, ketal, hydrazone, semicarbazone and enol groups, in any position of the naphthalene or indene structure, or also halogen atoms or epoxy groups. The starting materials can also con tain aliphatic side chains, for example substituted or unsubstituted alkyl radicals, such as methyl, ethyl, propyl or isopropyl radicals or also methyl, ethyl or propyl radicals substituted for example, by free or functionally converted hydroxyl, oxo or carboxyl groups. Specific starting materials include dzl-8-oxo-decalin, d:l-3:8-dioxo-decalin, d:l-8-oxo-9-methyl decalin, dzl- 3 8-dioxo-di-methyl-decalin, d l-3-hydroxy8-oxo9-methyl-decalin, d,l-A -3-oxo-0ctalin, d:l-A -3 S-dioxooctalin, d:l-A -3:8-dioxo-9-methyl-octalin and its enol derivatives, as for example d:l-A -3-alkoxy or acyloxy-S-oxo-9-methyl-hexahydronaphthalenes, dzl-A 3:8-dioxo-9-methyl-4-carboxymethyl-, carboxyethyl-, carboxypropyl-octalins and their functional derivatives, d:l-A -3-oxo-4:7-dihydroxy-9-methyl-octalin, d:l-A -3- oxo-6:7-isopropylidene-dihydroxy-9-methy1-octalin, d:1- A -3-oXo-9-methyl-hexahydronaphthalene, dzl-oxooctahydro-indene, d:l-1-oxo-8-methyl-octahydro-indene, dzl-l-oxo--hydroxy-8-methyl-octahydro-indene, dzl l :5- dioxo-8-methyl-octahydro-indene, d:l-A -1 :5-dioxo-8- methyl-hexahydro-indene and its enol derivatives, as for example d:l-A -1-oxo-5-alkoxyor acyloxy-3-methyltetrahydro-indene, d:l-A -l :5-dioxo-8-methyl-4-carboxy- 'ice methyl-carboxyethel or carboxypropyl-hexahydro-indene and their functional derivatives.

I have made the surprising observation that in the reduction according to this invention the enantimorphous forms of the racemates mentioned are hydrogenated at the same or dilferent speed, depending on the constitution of the starting material. Derivatives of d,l-A 3,8-dioxo-9-methyl-octalin, when subjected to the action of the aforementioned enzymes, are converted into two diastereoisomeric hydrogenation products, that is to say, conversion of the two enantiomorphous forms proceeds practically at the same rate.

The resulting diastereoisomers have different physical properties and are therefore easy to separate from each other and to isolate in pure form.

Dilferences in the reaction rate of the twoenantiomorphous forms are observed, for example, withstarting materials derived form d,l-A -1,S-dioxo-S-methyl-hexahydro-indene.

below.

Racemate 4 OH OH l O=c 0'- l l o o II J Racemate l on 0 It is advantageously carried out in such a manner that the culture of a micro-organism is caused to act upon the starting material. According to the micro-organism In these cases practically only one enantiomorphous form of the starting material is reduced, 1

used there are obtained, for 'exampleytwo' diastereomerie compounds inwhich one. oxo group or carbon-carbon double bond is saturated with hydrogen. The process can, however, be carried out in such a "manner that "ii! one operation several culturesare eausato 'ac 't 'u'pbn the starting material, it havin'g been proved advanta'geous in sucha case'to carry-out the ation of the individual -be incubated with cultures of the aforesaid micr'o-or ganisrnsunder knownanaerobic conditions, or even aerobic conditions. The growth may becarried out in 'a' surface 'culture or, advantageously, a submerged culture,:'the material being agitated or stirred.

The cultures contain assimilable carbon, for example carbohydrates, andif desired growth-promoting substances, for example, cornsteep liqu'or or beer wort, andinor'ganic salts. Acc'ord ingly, 7 natural, synthetic or semi-synthetic nutrient solutions: may .be .used.

The following gives a description of the simplest method of carrying out the process, but the invention is not limited thereto: The organisms are cultured in apparatus and-"under conditions similar 'to' those known as the so-called deep tank method in the production of antibiotics. After developing the culture, the starting material is added in the form of afiri'e 'dispersion or solution, "rd-reins ate in methanol, acetone or ethylene glycol, and incubation is continued. Finally the mycelium is separated, the filtrate and/or the mycelium mass is extracted, 'tandthe d-form and/or thel-form of the hydrogenation .products. and/or anyfienantiomorphous form of the starting material is isolated from theextract in known manner, for example, by a demixing method, adsorption, chromatography, crystallisation, conversion into functional derivatives such as Girard compounds or the like. The reactions may also be carried out by first separating the active enzymes partly or completely from the corresponding cultures of the aforesaid organisms and using them in the absence of the growing cultures. Thus, for exampley the mycelium torm'edby the culture of the organism is separated, suspended in water or a buffer, solution, and then the starting material is added to the resuIting suspension and incubated. It is also' possible to extractthe enzymes from the cultures and to use the resulting "extracts for the reactions.

If it is -desiredfio use several micro-organisms in one operation, the procedure may be, for? example, as follows: After developing athe culture of the first organism the starting material is added indie form? of a fine dis persion or solution, for example, in methanol, acetone or ethylene glycol, and incubation is continued until the maximum reaction has been attained; Then, without previousi filtratioh or isolation ethic-reaction product, 1

in the 'yrithe'sis" brzspaean tive steroids, fo 'ex'a'mpie, 'by'tiietliods" analogous" m thosealppliedtof the correspond- 4 -ing-racemic compounds. 'A-s a result of theclassical investigations of Pasteur a microbiological method for recovering an antipode from racemates has occasionally been used. Ordinary fungi or bacteria have been used for this purpose, which assimilate the starting materials, the natural (d) antipodes being assimilated more rapidly than the non-naturalil) antipodes. In theseprocesses, therefore, in order to obtain an optically pure product, the microbiological treatment must be continued until at least one form, and usually the biologically more interesting form, hasbeencmnpletely'destmyed. In contradistinction thereto, the new processyields even with only partial reaction the hydrogenation products in an optically pure form. t

The following examples illustrate the invention:

Example 1 In a shaking vessel, 40 grams of cane sugar, 40 grams of Difco tryptone, '8 grams of sodium nitrate, 4 grams of dipotassium phosphate, 2 grams of magnesium sulphate, 2 gramsof potassium-chloride and 0.04 grams of ferrous sulphate are dissolved in 4 liters of water, adjusted to pH 7, treated with 10 grams of calcium carbonate, sterilised and inoculated with a culture of Curvalaria falcata. After 3 days shaking at 27 C. a solution is added of 1 gram of d:l-A -3:8-dioxo-9-methyl-octalin inlS cc. of acetone and shaking is continued. After 3 days, the-mycelium is separated and washed with water andethyl acetate. The combined filtrates are extracted by shaking with ethyl acetate and the extracts washed with .diulte hydrochloric acid, potassium bicarbonate "solution and water dried and evaporated under vacuum. The paper chromatographic examination of the residue (1.6 grams) shows the presence of A* -3-oxo-8 -hydroxy- 9-methy1-octalin and a little starting material. The residue 'is chromatographed on grams of alumina by the fractional elution method, elution beingcarried outwith benzene, benzene-ether'mixtures, 'e'ther and ether-ethyl acetate mixtures. The paper chromatographic examination of'the individual'fractions (each 80 cc.) shows that thefirst benzene fractions contain starting material which is optically inactive. In the benzene. fractions "5 and'6 there is anegatively rotating oil,-which with "p-nitrobenzoyl' chloride in pyridine is convertedinto its 'p-nitro- "Fractions '7 23 eluted withbe'nzenecontain an oil which "possesses a'positive opticalrotatio'nand constitutes 2S 3 oxo-8-hydroxy 9-methyloctalin. With ether-arid "with ether-ethyl acetate mixtures a "strongly negatively .r'otat'ing, crystalline substance is eluted which is recrystallized from ether-petroleum ether mixtures (M. 'P. "94 95 C. [o =-l29) and which constitutes A 3 oxo 8 hydroxy-9-methyl-octalin. Analysis?- Found; C=73.00%, H=9.06%; ultra-violet adsorption spectru'rn in absolute alcoholrma'x. 240m (lOg e=4.l5); 'p-nitrobenzoate: MVP. 122.5" C., [a] =+87.

The oily (+)-A -3 oxo*S-hydroxy 9-methyl-octalin formsa crystalline p nitrobenzoate: M. P. 1-95 C., [1], -+159. By saponification by means-of methanolic caustic potash solution, the free-alcohol is reformed. :Itis distilled under high vacuum: [a] =-+203, ultra-violet adsorption spectrum in absolute alcohol: max. 240 m/n (log e=4.15).,

Example 2 ture-of Ophiob 0 lus herpotrichus. Shaking is carried out for 3 'daysat 27 C. and then under sterile conditions a solution added of-J gram of d:l-gA -3:8-dioxo-9 methylsame temperature. After 3 days, the mycelium is separated and washed with water and ethyl acetate. The combined filtrates are shaken with ethyl acetate and the extracts washed with dilute hydrochloric acid, potassium bicarbonate solution and water, dried and evaporated under vacuum. Paper chromatographic examination of the residue (1.01 grams) shows the presence of A -3- oxo-8-hydroxy-9-methyl-octalin and of starting material. The residue is worked up as described in Example 1. There is obtained, together with a little optically inactive starting material, the crystalline ()-A -3-oxo-8-hydroxy-9-methyl-octalin (M. P. 94-95 C., [a] =l2 9) and the oily (+)-A -3-oxo-8-hydroxy-9-methyl-octalin ([oc] =-l-203).

Example 3 The nutrient solution describedin Example 2 is inoculated with a culture of Rhizopus nigricans, instead of Ophiobolus herpotrichus and dzl A 3:8 diketo 9 methyl-octalin is incubated in thesame manner. After extraction and working up in an analogous manner, there is obtained, in addition to a little starting material, crystalline -A -3-oxo-8-hydroxy-9-methyl-octalin of M. P. 94-95 C., [a] =-129 and the oily (+)-A -3- oxo-8-hydroxy-9-methyl-octalin [a] +203 Example 4 In a shaking vessel a solution of 40 grams of crude glucose, 20 grams of peptone, grams of meat extract (Oxo Lab. Lemco) and gramsof sodium chloride in 4 liters of water is mixed with 40 grams of calcium carbonate, the pH value adjusted to 7.5, the mixture is sterilised and then inoculated with a culture of Streptomyces coelicolor. After agitation for 4 days at 27 C., there is added, as described in Example 1, a solution of 1 gram of d:1-A -3:8-dioxo-9-methyl-octalin in 15 cc. of acetone and agitation is continuedat 27 C. After 10 days, working up and chromatography are carried out as described in Example 1. There is obtained, in addition to a little starting material, the crystaline (-)-A -3- oxo-8-hydroxy-9-methyl-octalin of M. P. 9495 C., [a] =126 and the oily (l)-A -3eoxo-8-hydroxy-9- methyl-octalin ([a] =+203).

Example 5 A solution of 0.25 gram of (-)-A -3-oxo-8-hydroxy- 9-methyl-octalin in 4 cc. of pyridine is treated with 4 cc. of a pyridine-chromium oxide complex suspension, corresponding to 0.340 gram of chromiumtrioxide. The mixture is allowed to stand at room temperature and after 2 days 20 cc. of water are added. The solution is then concentrated under vacuum at C. and extracted by shaking with a benzene-ether mixture. The extracts are washed until neutral, dried and evaporated. 'The residue is chromatographed on 8 grams of alumina by the fractional elution method, elution being carried out with benzene-petroleum ether mixtures, benzene,benzene-ether mixtures and with ether. and the benzene eluates contain ()-A -3:8-dioxo-9- methyl-octalin which is crystallised from an ether-petroleum ether mixture, M. P. 50.5 C., [a] =-100, ultraviolet absorption spectrum in absolute alcohol; A max. 244 m (log e=4.10). The benzene-ether and ether fractions contain also some starting material.

Example 6 250 mg. of (+)-A -3-oxo-8-hydroxy-9-methyl-octalin are oxidised as described in Example 5 with pyridinechromium oxide complex, worked up and chromatographicallypurified. There is obtained together with some starting material, the (4-)-A -3:8-dioxo-9-methyloctalin which is recrystallised from an ether-petroleum ether mixture, M. P. 50 C., [a] =+100, ultra-violet absorption spectrum in absolute alcohol: A max. 244 mp (log i=4-10).

The benzene-petroleum ether 6 Example 7 secondary potassium phosphate, 2 grams of magnesium sulfate, and 0.04 gram of ferrosulfate are dissolved in 4 liters of water, the pH adjusted to 7, the solution mixed with 10 grams of calcium carbonate, sterilized, and inoculated with a culture of Curvalaria falcata. The culture is shaken for 36 hours at 27 C. after which a solution of 0.430 gram of d,l-A -l,S-dioxo-8 -methylhexahydro-in dene in 10 cc. of acetone is added under sterile conditions, and shaking continued at 27 C. After 3 days the mycelium is separated and washed with acetone and ethyl acetate. The combined filtrates ar exhaustively extracted with ethyl acetate. The extracts are washed with dilute hydrochloric acid, sodium bicarbonate solution and water, dried and evaporated under reduced pressure. The residue is chromatographed on grams of alumina by the fractional elution method with the use of benzene-ether mixtures, ether, ether-ethyl acetate mixtures, ethyl-acetate, and methanol. The individual fractions (200 cc. each) are evaporated in vacuo and investigated by paper chromatogv raphy (propylene glycol-toluene system). The first two benzene fractions contain only oily impurities, whereas the other benzene fractions contain a substance which in the paper ehromatogram behaves like the starting material (R =0.68). These fractions are combined and distilled in a high vacuum. From an ether-petroleum ether mixture crystals are obtained which melt at 58-60 C., [a] -312 (in benzene). The U. V. and I. R. adsorption spectra are identical with those of the starting material. The substance thus is the l-A -1,5-dioxo-8- methyl-hexahydro-indene.

The portions eluated with benzene-ether mixtures (19:1 and 9: 1) consist of d-A -5-oxo-1-hydroxy-8-methyl-hexahydro-indene which has an R value of 0.12 and is obtained inthe form of a dextrorotary oil absorbing U. V.- rays. With benzene-ether mixtures (4:1) small quantities ofa l-evorotary and U. V.-absorbing oil are eluated which in the paper chromatogram shows the same behavior and is the l-A -5-oxo-1-hydroxy-8-methyl-hexahydro-indene. The fractions eluated with ether, etherethyl acetate mixtures, ethylacetate, and methanol show no optical activity.

Example 8 In an Erlenmeyer flask 100 cc. of beer wort are sterilized and inoculated with a culture of Rhizopus nigricans. After 24 hours shaking at 25 C. there is added under sterile conditions a solution of 0.03 gram of d,l-A -1,5 dioxo-8 methyl-hexahydro-indene in 1.5 cc. of acetone. Shaking is continued for 5 days at 25 C. and the culture then extracted with ethyl acetate. Paper-chromatographic examination of the extraction residue shows the presence of l A 1,5 dioxo 8 methyl hexahydro indene '(R -0.68), d A 5 oxo 1 hydroxy 8 methylhexahydro-indene (R =0.12), and a further U. V.-absorbing substance of R =0.05

Example 9 In an Erlenmeyer flask 100 m3 of a sterile nutrient solution containing per liter of tap water 20 grams of peptone, 5 cc. of corn steep liquor and 50 grams of glucose are inoculated with a culture of Ophiobolus herpotrz'chas. After 24 hours shaking at 25 C. there is added under sterile conditions a solution of 0.03 gram of d,l-A -1,5-dioxo-8- methyl-hexahydro-indene in 1.5 cc. of acetone. Shaking is continued for 5 days at 25 C. and the culture then extracted with ethyl acetate. Chromatographic examination of the extraction residue shows the presence of LA- 1,S-dioxo-8-methyl-hexahydro-indene (R =0.68), d-A 5-oxo1-hydroxy-8-methyl-hexahydro-indene (R =0.12) and a further U. V.-absorbing substance of R =0.05.

, Exampl 10" In anErlenmcyerfiask' 100 cc. of asterile'nutrient solutioncontaining per-liter-of tapwater 10 grams of-glu' cose,,.5l gramsof peptone, 3 gramsofmeatextract (x0 Lab: Lemco), grams ofsodiumchloride, and grams inoculated with acultureof After 24 hours shaking at C. there is added; under sterile conditions a solution ofjcalcium carbonate are Streptomyces; aoelicolor.

of (102w gram of d;l A" 1;5' dioxo- 8 methylheXahydrZo-indene-in 1.5 cc. of acetone: Shaking is continued for 5 days at 25 C; and the culture-then extracted with I ethyl acetate.

ofthe' extraction residue shows the presence jof l-A -1,5-

1'. Processfor splitting racematcs; which comprises subjectingracemates of bicyclic-alic'ycli'c compounds containing at leastone grouping reducible with the formation of a new centre 'ofasymmetryrto the action ofreducing enzymes produced by cultures of microorganisms, isolating at least oneofthe resulting optically active products and-subjecting'any hydrogenated product obtainedto the action of a dehydrogenating agent.

2.; Process according to claim 1 wherein cultures of microorganisms capable of reducing oxo groupsto hy droxy groups are used;

- methyl heitahydro indene' 3; Process according to claim 1, wherein cultures of microorganisms capable of redticing'a carbon-to-carbon' double bond are used:i

4: Process according to claim 1, wherein several micro organisms are, used in the same operation.

5. Process according to claim 1, wherein racemates of the hydronaplithalene series are used as starting material;

6-; Processfor splitting racemates which-comprises'subjectingra: member, selected from the'group consisting-of a, racemic dihydro-, tetrahydro hexahydro-, octahydro and. decahydro-naphthalene compound containing as 'reduciblegrouping at carbon doublebond, at least oneof which being of such nature 'that -a new centre of asymmetry is-formedon its saturation, to the action of reducing enzymes-produced=by cultures of microorganisms, ;1solating at least one of the resulting optically active products and subjecting any hydrogenatedproduct obtained to the action of adehydro genatingagent.

, 7'. Process accordingto-claim 6, wherein d,l,-8,-oxo,-de

15. Process according to'clairn 1, wherein racemates' offthei hydro-indeneseries are used as starting material.

16.- Process for splittingracemates which comprises subjecting a member selected from the group consisting of a racemicdehydro-, tetrahydro-, hexa'hydroand -'octahydroindene compound containing as reducible grouping Paper-chromatographic examination least one member selected from-theg group consisting of a carbon-to-oxygen and-a carbon-to at least one member selected fromthegroupconsistingof a carbon-to-carbgodand a carbon-to-oxygen double bond; at least: one, of 'which being; of such nature that a new centre of asymmetry is-formed omits-saturation, to the action of "reducing en zymes produced" by cultures'of isolating at" least one of the resulting;-

microorganisms, optically active products and subjecting-anyhydrogenated product obtained to. the action of a dehydrogenating agent- 1 r r 17'. Process according to claim 16; wherein the d,'l- A -l,S-dicxo-8-methy1:heXahydro-indene is used" as-starting. material;

18; Process according to claim 1, whereina culture of- Saccharomyces cerevisiae is used. 19. Process according to claim Bacterium putryificus; is.=usedl 1, wherein a culture of 20. Process.accordingtto claim ,1, wherein a culture, of actinomyceteyselected from the. groups cousistingoi the i 1 species Streptomycestlavendulae:and Streptomyccs cuelicolor is used; t

21. Process according-tonclaim 1, wherein amculture of,

Cur-vulania falcata =is-g used-.

22. Process accordinglto claim 1-, ,wherein a, culture of,

fungi selectedlfrom the: groupconsisting of the species Rhizopus nigricans and Rhizopus arrhizus is used.

23., Process accordingtoclaim 1, wherein a culture of Ophiobolus .herpotriclrusl isiusedt.

24. Processtfor splitting: racemate; which comprises subjecting racemates Lot rbicyclic-alicyclicj compounds containingtat least: oneagrouping, reducible; with the formationnofrarnewncentre of asymmetry,t to theaction of,re,-

ducing enzymes :producedwbycultures 10f microorganisms, and isolating-at: least;- one. of the resulting opticallyt active,

products.

25: Process fort sp itting racemates which comprises subjecting a member selected; from .thegroup consisting,

of a racemic dihydro-,,tetrahydro.-, hexahydro, octahydroand decahydroenaphthalene compound containing as 1 reducible grouping at least one. member. selected from the group consisting of: a carbon-,to-oxygen and a carbom to-car-bom double bond; atle'ast onehof which, being-of such nature that a new centre ofiasymmetryis formed on:

its saturation, to the action ofreducing enzymes produced by cultures of microorganisms, and isolating at least oneof;the resulting-opticallyractive products,

1 26-. Processionsplitting; racernates; whichcomprises subjecting a memberg-selected from the groupwonsisting ot1a racemic dihydroratetrahydro hexahydro-, and octahydro-indene; compound containing, a reducible g p at least one;membernselcctedirom the group, consisting, of a carbomto-carbortt and, a, carbon-toroxygfinn double, bond; at least one. 0frwhich1heingno suchEnature that a new centre. of asymmetryfisformfid -0n-,its, saturation, to. the actiorrtofred'ucingenzymes produced by cultures'of microorganisms; anduisolatingt at 'least' one of the resulting optically activenproducts.

Referencest Cited-in; the tile, of: this patent UNITED STATES PATENTS 2,186,906 Mamolif nu; Ian. 9, 1940 2,341,110 Mamoli' Feb. 8, 1944 2,511,867 Newberget al.' June 20, 1950 2,602,769 Murray July 8, 1952 2,616,828 Levintow ett-al, Nov. 4, 1952 2,658,023 Shull u Nov. 3, 1953 2,778,776 wettstcin Jan. 22, 1957 OTHER REFERENCES Karrerv Organ icC-hemistry, 4th English Edition, Elsevir-Pub. Co., Inc, 1950, page 103. 

1. PROCESS FOR SPLITTING RACEMATES, WHICH COMPRISES SUBJECTING RACEMATES OF BICYCLIC-ALICYCLIC COMPOUNDS CONTAINING AT LEAST ONE GROUPING REDUCIBLE WITH THE FORMATION OF A NEW CENTRE OF ASYMMETRY, TO THE ACTION OF REDUCING ENZYMES PRODUCED BY CULTURES OF MICROORGANISMS, ISOLATING AT LEAST ONE OF THE RESULTING OPTICALLY ACTIVE PRODUCTS AND SUBJECTING ANY HYDROGENATED PRODUCT OBTAINED TO THE ACTION OF A DEHYDROGENATING AGENT. 